Use of leptin antagonists for treating insulin resistance in type II diabetes

ABSTRACT

The invention relates to pharmaceutical agents containing leptin antagonists for treating Type II diabetes. One leptin antagonist is based on a murine leptin fragment and comprises amino acids 116 to 167 or 116 to 166. Methods of treating Type II diabetes are also disclosed.

BACKGROUND OF THE INVENTION

The present invention relates to the use of leptin antagonists fortreating insulin resistance in Type II diabetes and to a pharmaceuticalfor treating such resistance.

Diabetes is one of the most frequently occurring metabolic diseases inindustrialized countries. There are some 110 million diabeticsworld-wide; while approx. 10 million of these are Type I diabetics, theoverwhelming majority (approx. 100 million) are Type II diabetics. Thedisease is caused by faulty regulation of glucose metabolism. In Type Idiabetes, failure of the β cells in the pancreas results in insulin nolonger being formed. This lack of insulin leads to an increase in bloodglucose and, if not treated by supplying insulin, to ketoacidosis,diabetic coma and death of the patient. In Type II diabetics, the causalrelationships are different and are characterized by the initialdevelopment of insulin resistance, i.e. diminution in the ability of thecells to respond adequately to insulin. Excessive weight and lack ofphysical activity, in particular, are regarded as being responsible forinducing insulin resistance. The latter condition is not noticedinitially since it is offset by an increased secretion of insulin.However, the continuing insulin resistance leads, in a process extendingover many years, to failure of the endogenous compensation mechanism andconsequent development of Type II diabetes. While diet and physicalactivity can delay this sequence of events, they are frequently unableto prevent manifestation of the disease. Medicinal intervention is thenrequired in order to control the blood glucose adequately.

It is of crucial importance for the long-term success of the therapythat the blood glucose be maintained as narrowly as possible within thephysiological range. It is the current view that glucose levels whichhave been elevated for decades, as are found in poorly controlleddiabetics (both Type I and Type II diabetics), make an importantcontribution to late complications in diabetes. These late complicationsconstitute, in particular, blood vessel damage which leads to kidneydiseases, loss of sight and cardiovascular diseases. This so-called latedamage is an important factor contributing to mortality in diabetics.

In 1994, a new hormone, leptin, was described which is formed in fatcells and which is lacking in genetically overweight mice (ob/ob mice)(Zhang, Y., Proenca, R., Maffei, M., Barone, M., Leopold, L., andFriedman, J. M. (1994). Positional cloning of the mouse obese gene andits human homologue. Nature 372, 425-432.). Human leptin and murineleptin are to a large extent identical. Injecting ob/ob mice withrecombinantly prepared leptin leads to a reduction in nutrient intakeand to a decrease in weight (Pelleymounter, M. A., Cullen, M. J., Baker,M. B., Hecht, R., Winters, D., Boone, T., and Collins, F. (1995).Effects of the obese gene product on body weight regulation in ob/obmice. Science 269, 540-543.). There has so far been no indication thatmutations in the ob gene might be responsible for the frequentoccurrence of obesity in humans (approx. 30% of the population ismarkedly overweight in the USA). Systematic investigations havedemonstrated that serum levels of leptin are increased in obese humansas they are in various animal models of obesity (Dagogo-Jack, S.,Fanelli, C., Paramore,: D., Brothers, J., and Landt, M. (1996). Plasmaleptin and insulin relationships in obese and nonobese humans. Diabetes45, 695-698; Considine, R. V., Sinha, M. K., Heiman, M. L., Kriauciunas,A., Stephens, T. W., Nyce, M. R., Ohannesian, J. P., Marco, C. C.,McKee, L. J., Bauer, T. L., and Caro, J. F. (1996). Serum immunoreactiveleptin concentrations in normal-weight and obese humans. N. Engl. J.Med. 334, 292-295.). For this reason, it is assumed that leptin is afeedback signal which informs the brain of the quantity of energy whichis stored in the fat tissue. According to this assumption, it is thenthe function of the brain to decrease feed intake by inhibitingappetite, on the one hand, and to stimulate basal metabolism on theother. In human obesity, this regulatory circuit appears to beinterrupted.

In addition to this, it is assumed that leptin also acts directly ontissues outside the brain.

Three studies relating to the direct effect of leptin on isolated cellshave so far been published:

Kroder et al. (Kroder, G., Kellerer, M., and Häring, H. (1996) Exp.Crin. Endocrin. Diabetes 104 Suppl. 2, 66 (Abstract)) made theassumption that leptin establishes a connection between insulinresistance and obesity and report that leptin decreases insulin-inducedphosphorylation of the insulin receptor and of insulin receptorsubstrate 1 (IRS-1) in rat 1 fibroblasts which are overexpressing thehuman insulin receptor. The extent to which leptin also exerts aninfluence on the end points of the insulin effect, for examplestimulation of glucose transport or glycogen synthase, was notinvestigated or discussed.

It has been demonstrated that sensitivity to lipogenic hormones(dexamethasone and insulin) is decreased in transformed 30A5preadipocytes which are overexpressing leptin (Bai, Y. L., Zhang, S. Y.,Kim, K. S., Lee, J. K., and Kim, K. H. (1996) J. Biol. Chem. 271,13939-13942). Fatty acid synthesis and the synthesis of neutral lipidswere decreased by leptin overexpression even in the non-stimulatedstate. While control cells exhibited a marked increase in the rate oflipid synthesis after the cells had been treated With dexamethasone orinsulin, or a combination of the two hormones, cells which wereoverexpressing leptin were hardly stimulated at all under thesecircumstances. In addition to this, an investigation was also undertakenof the inhibition of glycerophosphate dehydrogenase activity and ofacetyl CoA carboxylase expression which occurs after treating the cellswith a combination of dexamethasone and insulin. It was found that itwas not possible to stimulate leptin-expressing cells. The effects whichwere observed indicate that leptin suppresses lipid metabolism in ageneral manner. There is no mention of any possible connection betweenobesity and insulin resistance.

In another model system, i.e. C₂C₁₂ mouse myotubes, it was found thatleptin exhibits insulin-like effects (Berti, L., Kellerer, M., andHäring, H. (1996) Diabetologia 39 Suppl. 1, A59 (Abstract)). This studyreports that both glucose transport and glycogen synthesis arestimulated by leptin. These findings conflict with those reported by theother authors and the results which are presented here. They possiblyinvolve an effect which is specific for this cell type.

In our investigations into the effect of leptin on isolated ratadipocytes, a model system for fat tissue, it has now been found,surprisingly, that the insulin sensitivity of important metabolicpathways of the fat cell such as stimulation of lipogenesis, of glucosetransport and of glycogenesis, is drastically reduced (Example 4)whereas the basal values remain unaffected. The same applies to theinhibition of isoproterenol-stimulated lipolysis. Glucose transport inisolated rat adipocytes is stimulated approximately 14-fold by addinginsulin (10 nM). This capacity to be stimulated is reduced in adose-dependent manner by preincubating with leptin at differentconcentrations for 15 hours. Leptin desensitizes the cells, i.e. aninsulin resistance is produced. Dose/effect curves for insulin atdifferent leptin concentrations (Example 5) demonstrate that theconcentrations at which effects can already be detected in vitro, bothas regards insulin (0.1-0.2 nM) and as regards leptin (0.5-1 nM), arewithin the physiological range (Dagogo-Jack et al., 1996; Considine etal., 1996). Higher leptin levels (2-4 nM) (Dagogo-Jack et al., 1996;Considine et al., 1996), are found in obese humans, ,so that it ispossible that the insulin effect is more strongly impaired in thesesubjects. The conclusion therefore suggests itself that chronicallyelevated leptin, as can be seen in obese subjects, leads to insulinresistance. As already explained above, insulin resistance is animportant factor in the pathogenesis of Type II diabetes.

It is therefore an object of the invention to provide novel leptinantagonists that may be formulated in pharmaceutical compositions. It isanother object of the invention to provide methods of treating Type IIdiabetes and other insulin-related disorders.

The invention consequently relates to the use of leptin antagonists, inparticular those which are derived from leptin itself, for preparing apharmaceutical for use in Type II diabetes. The leptin antagonists forthis use are described in more detail below.

SUMMARY OF THE INVENTION

According to a first object of the invention, pharmaceuticalcompositions are provided which comprise an antagonist of leptin.According to this same object, pharmaceutical compositions are disclosedwhich comprise leptin antagonsists which are derived from leptin.Further according to this object, pharmaceutical compositions arerevealed which comprise a leptin antagonist that is a soluble leptinreceptor, or a derivative thereof.

According to a second object of the invention, methods are providedwhich utilize the inventive pharmaceutical compositions in the treatmentof Type II diabetes. Also according to this object, methods are providedfor restoring or amplifying the physiological effect of insulin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the amino acid sequence of human leptin (SEQ ID NO: 4). Thetwo cysteins are linked by a disulfide bridge.

FIG. 2 is the amino acid sequence of murine leptin (SEQ ID NO: 5). Thetwo cysteins are linked by a disulfide bridge.

FIG. 3 is the amino acid sequence of fragment 116-167 of murine leptinSEQ ID NO: 6). The two cysteins are linked by a disulfide bridge.

DETAILED DESCRIPTION OF THE INVENTION

The invention which is presented here is directed towards compositionsand methods for reducing, or completely eliminating, insulin resistanceby inhibiting the effect of leptin. For this purpose, use can be made ofpeptides which act as leptin antagonists and which lead, in isolated fatcells in vitro, to abolition of leptin-induced insulin resistance. Thesepeptides are consequently suitable for the therapy of insulinresistance, preferably in obese patients.

Leptin Antagonists

Leptin antagonists according to the invention specifically includepeptide antagonists. The said peptides are derived from leptin fragmentsand may be obtained, for example, by chemically or enzymically cleavingintact leptin (e.g. with lysyl endopeptidase, trypsin, endo-Arg C orcyanogen bromide) or be prepared by being expressed, directly or as afusion protein, in microorganisms. In connection with preparing thepeptides in microorganisms, there is no compulsion to rely on thepresence of natural cleavage sites when selecting the fragments whichare to be expressed. Human and animal leptin, for example rat, mouse,pig or humanoid ape leptin, are suitable for deriving the peptides.

One suitable exemplary peptide extends from amino acid 116 to amino acid167 or from amino acid 116 to amino acid 166 (FIG. 3, SEQ ID NO: 6) inaccordance with the sequence which has been published in Zhang et al.,(1994) (Examples 3 and 6). In Example 6, adipocytes were incubated forapprox. 15 hours in the presence of 10 nM leptin and differentconcentrations of the antagonistic 116-167 leptin fragment. The cellswere then stimulated with 5 nM insulin. In this experiment, it was foundthat increasing quantitites of the antagonist lead to restoration of thecapacity to be stimulated by inulin and no resistance develops in thepresence of high concentrations of the leptin antagonist. Thus, usingthese and similar assays, one skilled in the art readily may ascertainthe antagonistic properties of any leptin antagonist that would beuseful according to the present invention.

In addition to this, analogues of the antagonistic leptin fragments canalso be used in which one or more amino acids is/are replaced ordeleted. Preferable replacements are conservative amino acidsubstitutions. Such conservative substitutions include charged-charged,polar-polar and hydrophobic-hydrophobic amino acid substitutions. Forexample, one or more aspartate residues can be replaced by glutamateresidues and/or vice versa and/or one or more leucine residues can bereplaced by isoleucine residues and/or vice versa. Other substitutionsand deletions may rationally be made based on steric and structuralconsiderations, such as amino acid size and propensity for helix makingor breaking.

Molecular biological and biotechnological methods can be used to alterand optimize the antagonistic properties of the said peptides in aspecific manner. In addition to this, the peptides can be modifiedchemically, for example by means of acetylation, carbamoylation,formylation, biotinylation, acylation, or derivatization withpolyethylene glycol or hydrophilic polymers, in order to increase theirstability or modulate their plasma half-life and pharmacokinetics.

Antibodies against leptin, in particular their leptin-binding domains,are also suitable as leptin antagonists for the said purpose. Inaddition to this, soluble leptin receptors and/or leptin receptorfragments, and fusions thereof with other proteins (e.g. the IgG Fcregion), are also suitable. Like the peptide antagonists, any leptinantagonist which is a protein may be altered by molecular biologicalmeans. Similarly, these antagonists may be prepared by being expressed,directly or as a fusion protein, in microorganisms or any number ofstandard expression systems.

Pharmaceutical Compositions

The invention furthermore relates to a pharmaceutical which comprisesthe leptin antagonists which are described in this patent application.

The pharmaceuticals can be used, for example, in the form ofpharmaceutical preparations which can be administered orally, forexample in the form of tablets, coated tablets, hard or soft gelatincapsules, solutions, emulsions or suspensions. They can also beadministered rectally, for example in the form of suppositories, orparenterally, for example in the form of injection solutions. Thepharmaceuticals can also be administered by way of the mucous membranesof the nose, of the mouth or of the lung. In order to producepharmaceutical preparations, these compounds can be worked intotherapeutically inert, organic and inorganic excipients. Lactose, cornstarch or derivatives thereof, talc and stearic acid or salts thereofare examples of such excipients for tablets, coated tablets and hardgelatin capsules. Water, polyols, sucrose, invert sugar and glucose aresuitable excipients for preparing solutions. Water, alcohols, polyols,glycerol and vegetable oils are suitable excipients for injectionsolutions. Vegetable and hardened oils, waxes, fats and semi-liquidpolyols are suitable excipients for suppositories. The pharmaceuticalpreparations can also comprise preservatives, solvents, stabilizers,wetting agents, emulsifiers, sweeteners, dyes, flavorants, salts foraltering the osmotic pressure, buffers, coating agents, antioxidantsand, where appropriate, other therapeutic active compounds.

Oral administration and injections are preferred. For injection, thenovel leptin antagonists are formulated in a liquid solution, preferablyin a physiologically acceptable buffer such as Hank's solution orRinger's solution. However, the novel leptin antagonists can also beformulated in solid form and be dissolved or suspended prior to use.

Typical formulations contain a therapeutically beneficial amount of aleptin antagonist. A therapeutically beneficial amount may be the sameas a therapeutically effective amount, as discussed below. Additionally,a therapeutically beneficial amount may be a unit dose, which eitheralone or in multiples may be used to provide a therapeutically effectiveamount of leptin antagonist. Thus, a therapeutically beneficial amountwill rely, inter alia, on the nature of the disorder being treated.

Methods of Treatment

The methods of the invention are useful in treating any dirsorder inwhich the action of leptin is implicated. In view of the presentdislcosure and the observation that leptin inhibits some of thephysiological activities of insulin, the inventive methods areparticularly useful in the treatment of disorders involvingperturbations of insulin activity, and especially Type II diabetes. Suchperturbations in insulin activity include alterations in lipogenesis,glucose transport glycogenesis and lipolysis. Accordingly, the inventivemethods include methods for treating Type II diabetes and methods forrestoring or amplifying the physiological effects of insulin.

A typical method entails administering to a patient in need of treatmenta therapeutically effective amount of a leptin antagonist. A patientwill be in need of treatment when suffering from a disorder in which theaction of leptin is implicated. Treatment is especially indicated when apatient is suffering from Type II diabetes. Treatment is also indicatedwhen a patient is suffering from a disorder characterized by aperturbation in insulin activity, such as alterations in lipogenesis,glucose transport, glycogenesis and lipolysis. In disorders where such aperturbation is implicated, methods for restoring or amplifying thephysiological effect of insulin are useful.

A therapeutically effective amount will depend, for example, on thenature of the disorder being treated, the route of administration, theparticular characteristics of the antagonist chosen, and especially thejudgement of the attending clinician. A therapeutically effective amountgenerally is an amount sufficient to effect treatment of the diseasetargeted or to accomplish the stated goal of the method, for example,restoring or amplifying the physiological effect of insulin. Ultimatelythe therapeutically effective amount will depend on the clinicallydetermined efficacy and toxicity of each leptin antagonist. Suchdeterminations are routinely made and well within the ordinary skill ofthe clinician.

The term “treating” in its various grammatical forms in relation to thepresent invention refers to preventing, curing, reversing, attenuating,alleviating, minimizing, suppressing or halting the deleterious effectsof a disease state, disease progression, disease causative agent orother abnormal condition.

The doses which are preferred for systemic administration are fromapprox. 0.01 mg/kg to approximately 50 mg/kg of body weight and per day.

The invention is clarified below by means of the Tables and Exampleswithout being restricted thereto.

EXAMPLES Example 1 Cloning Murine Leptin

RNA isolation—Epididymal fat pads are removed from adult mice andshock-frozen in liquid nitrogen. 1 g of fat tissue is ground in a mortarunder liquid nitrogen, after which 15 ml of a 5 M solution ofguanidinium thiocyanate in 50 mM Tris (pH 7.5), 10 mM EDTA and 0.1 M DTTare added and the whole is homogenized vigorously in order to obtain afine dispersion. After the tissue particles have been completelydissipated, 10 g of solid CsCl are added and the mixture is stirred atroom temperature. After adding 10 ml of H₂O, 9 ml of a 5.7 M solution ofCsCl are overlaid with 25 ml of this solution in a centrifuge tube.After centrifuging for 15 hours in an SW28 rotor at 25,000 rpm (18° C.),the tube is deep-frozen in liquid nitrogen and the bottom quarter of thetube is cut off using a hot scalpel blade; the frozen contents are takenout and the RNA pellet is scraped off their bottom end. The RNA isdissolved and then precipitated with ethanol.

cDNA synthesis—In a mixture, 1 μg of total RNA from fat tissue and 1 μgof the specific primer oligonucleotide 5′-GAATGCAGAATAAATAAATA (SEQ IDNO.: 1; Zhang et al., 1994) are dissolved in 10 μl of H₂O and are thenheat-denatured and incubated at 65° C. for 5 min. After adding 0.5 μl ofRNase inhibitor, in each case 5 nmol of dNTP and 0.5 μl of AMV reversetranscriptase (Boehringer Mannheim), the mixture is incubated at 42° C.for 1 h. After that the cDNA is made up to 200 μl with H₂O and stored at−20° C.

PCR—3 μl of the specifically primed cDNA are amplified with 0.5 μg eachof the two primers 5′-GAAAGAAGGATCCAGTGCCTATCCAGAAAGTCCA (SEQ ID NO.: 2)and 5′-GGAGAGAAGCTTGAGGGAGAGAAATGAATGATGG (SEQ ID NO.: 3; Zhang et al.,1994) and 2.5 U of Taq polymerase (Perkin Elmer) for 30 cycles in thereaction buffer recommended by the manufacturer (1.5 mM MgCl₂, 200 μMdNTPs in 100 μl). Each cycle comprises 1 min at 94° C., 1 min at 55° C.and 2 min 72° C. Ligation—The specifically amplified PCR product (583bp) from a PCR preparation is cleaved, in each case at 37° C. for 2 hand under buffering conditions which accord with the manufacturer'sinstructions, with the restriction enzymes BamHI and HindIII (BoehringerMannheim), after which the 564 bp fragment is purified byelectrophoresis and isolated. It is incubated, at 30° C. for 2 h and in20 μl, together with 0.1 μg of BamHI- and HindIII-cleaved vector pQE31(Qiagen) and 20 U of T4 DNA ligase (New England Biolabs).

Cloning—5 μl of the ligation mixture are kept on ice for 30 min togetherwith 100 μl of transformation-competent E. coli cells of the HB101strain and the mixture is then swirled gently for 5 min in a water bathat 37° C. Following the addition of 0.9 ml of nutrient medium containing10 mM MgCl₂, the mixture is shaken at 37° C. for 1 h. 100 μl volumes ofthis mixture are in each case plated onto ampicillin-containing (100μg/ml) agar plates.

Identification of the clones—The clones which have grown overnight at37° C. are inoculated into 2 ml-volume liquid cultures containingampicillin, cultured to stationary phase and then centrifuged down. Thecells are suspended in 0.1 ml of 25 mM Tris (pH 8), 50 mM glucose, 10 mMEDTA and lysozyme (2 mg/ml) and, after having been incubated at roomtemperature for 5 min, are lysed by adding 0.2 ml of 0.2 M NaOH, 1% SDS.The chromosomal DNA is precipitated by adding 150 μl of 3 M Naacetate/acetic acid (pH 5.2) and centrifuged down for 5 min at 4° C.(10,000 rpm in a Sigma 2MK). The plasmid DNA is precipitated with 2.5volumes of ethanol, centrifuged down (see above) and, following anethanol wash, taken up in 100 μl of H₂O; 10 μl of RNase solution (10mg/ml) are then added.

The plasmid DNA is digested with restriction enzymes (BglI, XhoI+PvuII;Boehringer Mannheim) in accordance with the manufacturer's instructions,and the resulting DNA fragments are measured against marker DNA in anagarose gel following electrophoresis and staining with ethidiumbromide. Clones which have the correct fragment pattern are examined inthe same way using the restriction enzyme AfIIII (New England Biolabs)for the presence of the glutamine 49 residue.

The identity of in each case one E. coli clone containing Gln49(pQEob3-9) and one not containing Gln49 (pQEob3-4) was confirmed bymeans of DNA sequencing. The production of recombinant leptin containingthe presequence (SEQ ID NO.: 7) MetArgGlySer(His)6ThrAspPro (from thevector pQE31) followed by amino acids 22 to 167 from murine leptin waschecked in small cultures.

While both recombinant leptins (with and without Gln49) can be employedin the experiments which are described below, the examples specificallyrelate to leptin which contains Gln49 and which is obtained byexpressing pQEob3-9.

Example 2 Preparation of Leptin

Disruption—The bacteria from a 10 l fermentation are centrifuged at 4800rpm for 20 min. The sediment is frozen at −20° C. and subsequentlysuspended in lysis buffer (6 M guanidinium chloride, 0.1 M NaH₂PO₄, 10mM Tris/HCl, pH 8) (5 ml of lysis buffer/g of sediment), after which themixture is stirred at room temperature for one hour and then centrifugedat 4800 rpm for 30 min.

Ni-NTA chromatography—100 ml of Ni-NTA agarose FF (Qiagen, Hilden) areadded to crude extract which contains approx. 800 mg of leptin and thewhole is stirred at 4° C. overnight. The suspension is sucked through aglass column (ø5 cm) possessing a frit. The column, together with theNi-NTA agarose which is contained in it, is washed with 300 ml of lysisbuffer and then with 100 ml of lysis buffer containing 10 mM imidazole(5 ml/min). Fractional elution of the leptin then takes place byapplying a linear gradient of 10 to 200 mM imidazole in lysis buffer(gradient volume: 300 ml at 5 ml/min). The fractions are analyzed byRP-HPLC and those which are of adequate purity and concentration arecombined (=Ni-NTA eluate).

Folding—The Ni-NTA eluate is diluted with lysis buffer to aconcentration of 1-3 mg/ml and adjusted to pH 9 with sodium hydroxidesolution. After adding β-mercaptoethanol (from 4 to 6 mol ofβ-mercaptoethanol per mole of leptin), the mixture is incubated at roomtemperature for 2 hours in a sealed container. For reoxidation andrefolding, the solution is poured into 9 time's the volume of foldingbuffer (0.1 M Tris/HCl, pH 9) and the whole is stirred at 16° C. forfrom 16 to 24 hours while admitting air. Any turbidity which appears iscentrifuged off (4,000 rpm, 45 min).

Reversed phase HPLC—The folding mixture is adjusted to pH 3 with HCl andpumped onto a 2.5×30 cm RP column (PLRPS 300 Å, 10μ, PolymerLaboratories, Amherst, USA) at the rate of 20 ml/min. The column issubsequently washed with 300 ml of eluent A (0.1 % aqueous TFA). Leptinis eluted in 100 minutes (flow rate: 7 ml/min) by applying a gradient offrom 25 to 50% eluent B (0.09% TFA in acetonitrile). The fractions areanalyzed by means of analytical HPLC. Fractions of adequate purity andconcentration are combined (RP pool). The pool is treated with 7 mmol ofNa₂HPO₄/l and adjusted to pH 3 with NaOH, and the solvent is removed ona rotary evaporator. The aqueous leptin solution is then neutralizedwith NaOH (pH 7.4) and stored at 4° C. overnight. Any resultingturbidity is centrifuged off((4,000 rpm, 10 min).

Gel permeation chromatography—The neutralized and centrifuged leptinsolution is concentrated to 10-15 mg/ml by ultrafiltration and thensterilized by filtration. From 50 to 75 mg are loaded onto a Superdex 75column (2.6×60 cm, Pharmacia, Sweden). PBS (154 mM NaCl, 10 mM sodiumphosphate, pH 7.4) is used as the elution buffer, at a flow rate of 3ml/min. The leptin which has been purified in this way is then filteredonce again through a 0.22 μm membrane and stored at −70° C.

Example 3 Preparation of the Antagonistic 116-167 Fragment

1 ml of 1 M Tris/HCl, pH 8, is added to 40 ml of a solution of leptin (1mg/ml in PBS) and, after 160 μg of lysyl endopeptidase have been added,the leptin is digested at room temperature for 3 h. The mixture isadjusted to pH 3 and fractionated by RP-HPLC as described in Example 2.The 116-167 fragment is identified by electrospray mass spectrometry(5532 D), freed from solvent as described in Example 2, concentrated andpurified by means of gel permeation chromatography.

Example 4 Isolation of Adipocytes

Adipocytes were prepared from the epididymal fat tissue of male Wistarrats (140-160 g, Hoechst AG breeding station, Kastengrund) by means ofdigesting with collagenase (Rodbell, 1964, J. Biol. Chem. 239, 375-380),washed twice with KRH (25 mM Hepes free acid, 25 mM Hepes sodium salt,80 mM NaCl, 1 mM MgSO₄, 2 mM CaCl₂, 6 mM KCl, 1 mM sodium pyruvate, 0.5%BSA) and once with DMEM (Dulbecco's minimal essential medium),supplemented with 5.5 mM glucose, 20 mM Hepes (pH 7.4), 2% fetal calfserum, 1% BSA, 50 U of penicillin/ml, 10 mg of streptomycin/ml, by meansof flotation (800×g, 1 min, in small plastic tubes), and finally dilutedto a volume of 20 ml of DMEM/g wet weight of the fat tissue (cell titer:approximately 2.5×10⁵ cells/ml).

Example 5 Primary Culture of the Adipocytes and Incubation with Leptin

The adipocytes were incubated, at 37° C. for 15-18 h under an atmosphereof 5% CO₂ and with gentle shaking, in supplemented (see above) DMEM inthe presence of 100 nM phenylisopropyladenosine (4 ml of DMEM to 1 ml ofcells, cell titer approximately 5×10⁴ cell/ml, in 50 ml sterilepolypropylene: tubes) and in the presence or absence of leptin. Theadipocytes were subsequently washed three times with cold KRH andadjusted to a cell titer of approximately 3×10⁵ cells/ml by adding 0.7ml of glucose-free KRH to the cell layer which remained after thecomplete removal of the last washing solution. In order to determine thecapacity of the adipocytes to be stimulated by insulin, and theirsensitivity to insulin, following the primary culture, the washedadipocytes were incubated at 37° C. for 20 min in the absence orpresence of human insulin (0.02-50 nM final concentration), and glucosetransport or lipogenesis was then measured.

Example 6 Glucose Transport

Glucose transport was measured as the specific uptake of thenon-metabolizable glucose analog 2-deoxyglucose (Muller and Wied, 1993,Diabetes 42, 1852-1867). 50 μl of the adipocyte suspension in KRH, whichsuspension had or had not been preincubated with insulin (see above),were incubated, at 25° C. for 5 min, with 50 μl of KRH which wassupplemented with 2-deoxy-D-[2,6-³H]glucose (0.5 μCi, 0.2 mM). Theincubation mixtures were transferred to thin soft-plastic centrifugetubes, each of which already contained 200 μl of dinonyl phthalate oil,and immediately centrifuged (2,000×g, 30 sec). Using special shears, thetubes were severed within the oil layer (in the vicinity of the upperedge) and the upper halves of the tubes, together with the cell layerswhich had in each case been floated onto the oil layer, were transferredto scintillation vials. After 10 ml of scintillation fluid (water-based)had been added, the radioactivity associated with the cells wasmeasured. In order to correct for 2-deoxyglucose which had becomeenclosed in the cell interstices, or had diffused into the cells, in anon-specific manner, the radioactivity of cells which had beenpreincubated with cytochalasin B (20 μM) was subtracted from the totalcell-associated radioactivity of each individual incubation mixture(Gliemann et al., 1972, Biochim. Biophys. Acta 286, 1-9).

Example 7 Lipogenesis

Lipogenesis was measured as the incorporation of D-glucose intotoluene-extractable lipids (Moody et al., 1974, Horm. Metab. Res. 6,12-16). 200 μl of the adipocyte suspension in KRH were incubated inscintillation vials, at 37° C. for 20 min, in 680 μl of KRH which wassupplemented with 3.5 mM glucose and 20 μl of insulin solution.Lipogenesis was started by adding 100 μl of D-[3-³H]glucose (25 μCi/mlKRH). After incubating at 37° C., and gently shaking under an atmosphereof 5% CO₂, for 90 min, 10 ml of scintillation fluid (toluene-based) wereadded and the radioactivity in the toluene phase was determined aftershaking vigorously and subsequent phase separation (at least 4 h ofincubation). The radioactivity of the lipids in the toluene phase wascorrected for the radioactivity of an incubation mixture which containedthe same quantity of [³H]glucose but no cells.

Example 8 Inhibition by Leptin of Insulin-induced Glucose Transport andof Insulin-Induced Lipogenesis

Isolated rat adipocytes were incubated for 15 h in primary culture inthe presence or absence of increasing concentrations of leptin. Thecells were then washed and tested for glucose transport and lipogenesisin the basal and insulin (10 nM)-stimulated states. The stimulationfactor for insulin was calculated as the ratio betweeninsulin-stimulated and basal activities. Each value represents the meanfrom two independent adipocyte cultures with activity being determinedtwo or three times in each case.

TABLE 1 Insulin concentration: 5 nM Leptin Glucose Concentration [nM]Transport* Lipogenesis* 0 13.4 3.9 0.3 13.4 3.9 1 11.75 3.2 3 7.4 2.1510 3.5 1.5 30 2.35 1.15 100 1.5 1.05 *The factors are the quotients ofthe values following stimulation and the basal values.

The results are summarized in Table 1.

Example 9 Influence of Leptin on the Insulin Dose/Effect Curve

Isolated rat adipocytes were incubated for 16.5 h in primary culture inthe presence or absence of increasing concentrations of leptin. Thecells were then washed and tested for the stimulation of glucosetransport by differing concentrations of insulin. Glucose transportactivity is given as a “dpm value” of the 2deoxy-[³H]glucose which isspecifically associated with the cells. Each value represents the meanfrom two independent adipocyte cultures with the activity beingdetermined four times in each case.

TABLE 2 Measured parameter: glucose transport Leptin [nM] Insulin [nM] 00.05 1 2 5 10 30 100 0 671 654 634 688 712 755 688 747 0.02 784 735 678704 734 773 704 766 0.05 1285 1025 824 755 798 802 745 780 0.1 2406 16741189 860 883 856 789 803 0.2 4762 3320 1587 1006 923 941 852 813 0.56976 5138 2180 1377 1167 1209 943 883 1 7981 6834 3904 1916 1583 15731183 896 2 8576 7942 5510 2680 2140 2061 1374 1034 5 8956 8794 6985 43232950 2476 1782 1205 10 9064 9134 8241 6107 3682 2710 1972 1451 50 90729189 8932 7032 4031 2967 2114 1723

The value given is in each case the uptake of ³H-labeled 2-deoxyglucosemeasured in dpm (disintegrations per minute).

The results are summarized in Table 2.

Example 10 The Antagonism of Fragment 116-167 to the Leptin Effect

Isolated rat adipocytes were incubated for 17.5 h in primary culture inthe presence or absence of leptin (10 nM) and increasing concentrationsof leptin fragment 116-167. The cells were then washed and tested forstimulation of glucose transport or lipogenesis by 5 nM insulin. Thestimulation factor for insulin was calculated as the ratio between theinsulin-stimulated activity and the basal activity. Each valuerepresents the mean from two independent adipocyte cultures withactivity being determined three times in each case.

TABLE 3 Leptin: 10 nM Insulin: 5 nM Fragment Glucose Concentration [nM]Transport* Lipogenesis 0 3.55 1.5 0.3 3.65 1.65 1 4.5 2.15 3 5.75 2.6510 7.75 3.1 30 10.8 3.6 100 12.55 4 300 13.2 4 Insulin only 13.4 3.9*The factors are the quotients of the values following stimulation andthe basal values.

The results are summarized in Table 3.

FIG. 1: SEQ ID NO: 4

Human leptin 1-Met His Trp Gly Thr Leu Cys Gly Phe Leu Trp Leu Trp ProTyr   Leu Phe Tyr Val Gln Ala Val Pro Ile Gln Lys Val Gln Asp Asp   ThrLys Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp Ile   Ser His ThrGln Ser Val Ser Ser Lys Gln Lys Val Thr Gly Leu   Asp Phe Ile Pro GlyLeu His Pro Ile Leu Thr Leu Ser Lys Met   Asp Gln Thr Leu Ala Val TyrGln Gln Ile Leu Thr Ser Met Pro   Ser Arg Asn Val Ile Gln Ile Ser AsnAsp Leu Glu Asn Leu Arg   Asp Leu Leu His Val Leu Ala Phe Ser Lys SerCys His Leu Pro   Trp Ala Ser Gly Leu Glu Thr Leu Asp Ser Leu Gly GlyVal Leu   Glu Ala Ser Gly Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu  Gln Gly Ser Leu Gln Asp Met Leu Trp Gln Leu Asp Leu Ser Pro   GlyCys-167

FIG. 2: SEQ ID NO: 5

Murine leptin 1-Met Cys Trp Arg Pro Leu Cys Arg Phe Leu Trp Leu Trp SerTyr   Leu Ser Tyr Val Gln Ala Val Pro Ile Gln Lys Val Gln Asp Asp   ThrLys Thr Leu Ile Lys Thr Ile Val Thr Arg Ile Asn Asp Ile   Ser His ThrGln Ser Val Ser Ala Lys Gln Arg Val Thr Gly Leu   Asp Phe Ile Pro GlyLeu His Pro Ile Leu Ser Leu Ser Lys Met   Asp Gln Thr Leu Ala Val TyrGln Gln Val Leu Thr Ser Leu Pro   Ser Gln Asn Val Leu Gln Ile Ala AsnAsp Leu Glu Asn Leu Arg   Asp Leu Leu His Leu Leu Ala Phe Ser Lys SerCys Ser Leu Pro   Gln Thr Ser Gly Leu Gln Lys Pro Glu Ser Leu Asp GlyVal Leu   Glu Ala Ser Leu Tyr Ser Thr Glu Val Val Ala Leu Ser Arg Leu  Gln Gly Ser Leu Gln Asp Ile Leu Gln Gln Leu Asp Val Ser Pro   GluCys-167

FIG. 3: SEQ ID NO: 6

116-Ser Cys Ser Leu Pro Gln Thr Ser Gly Leu Gln     Lys Pro Glu Ser LeuAsp Gly Val Leu Glu Ala     Ser Leu Tyr Ser Thr Glu Val Val Ala Leu Ser    Arg Leu Gln Gly Ser Leu Gln Asp Ile Leu Gln     Gln Leu Asp Val SerPro Glu Cys-167

The two cysteines present in the sequences shown in Tables 4 to 6 arelinked by a disulfide bridge.

What is claimed is:
 1. A leptin antagonist which is a peptide having theamino acid sequence of SEQ ID NO.
 6. 2. The leptin antagonist of claim1, which in an in vitro assay, inhibits leptin from reducinginsulin-induced glucose transport levels in adipocyte cells in culture.